AT402890B - USE OF NOTOGINSENOSIDE R1 IN THE PRODUCTION OF MEDICINAL PRODUCTS - Google Patents

Description

AT 402 890 B

The invention relates to the use of notoginsenoside R1 (NR1) for the manufacture of medicaments.

In L. Zechmeister, Progress in the Chemistry of Organic Natural Products, Vol. 46, Springer-Verlag, Vienna New York 1984, pages 1-76, the chapter on " Saponins of Ginseng and Related Plants " the analysis and purification of saponins from ginseng and related plants, as well as the purification of Notoginseng-R1, whereby the property of Panax notoginseng as a tonic, haemostatic cum, coronary therapeutic and antihemorrhagicum is pointed out. It is stated specifically about Sanchi ginseng from Panax notoginseng that this ginseng, which is not present as a purified fraction, contains a hemostatic and thus has the exact opposite effect of increasing the fibrinolytic capacity. As far as the anti-hemorrhagic effect of Sanchi-Ginseng is discussed, it should be noted that this is caused by neurotoxin £ -N-oxal-L-a / 3-diaminoproproic acid.

Chemical Abstracts 119, 85 683 y discloses that total saponins from Panax notoginseng inhibit platelet aggregation in vitro and inhibit thrombosis in vivo by reducing the number of platelets. No effect of this saponin extract on the fibrinolytic system is described, but on the contrary mentioned that the fibrinogen level and the protrombin time remain unchanged. This is only an indication that the coagulation and fibrinolysis system is not affected.

The fibrinolytic system serves as a basic defense mechanism that controls the deposition of fibrin in both vascular and extravascular systems. Proper functioning of the fibrinolytic system is necessary to prevent hemorrhagic and thrombotic phenomena on the one hand, but also to prevent the formation of interstitial fibrin deposits and the subsequent scarring. The tissue plasminogen activator (t-PA) is believed to play an important role in triggering the (extrinsic) fibrinolytic cascade by converting the zymogen plasminogen to the active plasmin that breaks down fibrin. It is also believed that the fibrinolytic capacity of the plasma is significantly dependent on the concentration of circulating t-PA. The t-PA in plasma probably originates mainly from the vascular wall, where it is located in the endothelial cell. The urokinase plasminogen activator (u-PA) also plays a role in total fibrinolysis. It is believed that this plasminogen activator also comes at least in part from the vessel wall. The main inhibitor of fibrinolysis, plasminogen activator inhibitor 1 (PAI-1) is also synthesized by endothelial cells and there are data which show that the relative quantitative ratio between PAs and PAI-1 is important for the fibrinolytic capacity and thus for the prevention of thrombotic processes such as e.g. with myocardial infarction. The pharmacological regulation of the synthesis of t-PA, u-PA and PAI-1 is therefore useful to increase inadequate endogenous fibrinolysis.

Since both t-PA and PAI-1 are produced by endothelial cells, the regulation of their synthesis and secretion at the level of the endothelial cell is a quick and direct way of influencing the fibrinolytic potential of the blood. Recent studies have shown that the production of plasminogen activators and inhibitors in different cell types is regulated by a number of factors: The synthesis of t-PA in endothelial cells is affected by a variety of stimuli such as e.g. Thrombin, histamine, butyrate, retinoic acid and tumor promoters such as e.g. Phorbol-12-myristate-13-acetate (PMA) increased. Factors that regulate the expression of PAI-1 include lipopolysaccharides, thrombin, interleukin 1 (IL 1), tumor necrosis factor a (TNF a), transforming growth factor ß (TGF ß) and basic fibroblast growth factor (BFGF) and endothelial cell growth Supplement in combination with heparin. However, none of the substances listed above could be used successfully in vivo.

Bacterial sepsis caused by the release of bacterial endotoxins (LPS) is a life-threatening condition in which changes in coagulation and fibrinolysis caused by LPS cause intravascular clot formation and consequently organ failure. It is believed that LPS acts on endothelial cells by increasing their expression of tissue factor (TF) and PAI-1. Sufficient direct treatment of patients suffering from intravascular coagulation caused by LPS is currently not possible, and measures to treat the symptoms triggered by LPS, such as e.g. Hypercoagulation is limited on the one hand to heparin and on the other hand to the treatment of the causing bacterial sepsis with antibiotics. In China, the Chinese herbal drug Panax Notoginseng has been used for thousands of years by traditional Chinese doctors to relieve pain and treat stasis and cardiovascular diseases.

The use of NR1 obtained therefrom for increasing the fibrinolytic capacity and for directly inhibiting the LPS effects is described here for the first time. The invention now relates to the use of notoginsenoside R1 (NR1) for the production of medicaments for the treatment of conditions with reduced fibrinolytic activity or for the inhibition of endotoxin effects. Notoginsenoside R1 (NR1) is administered as a purified substance either intravenously or orally in the form of solutions or tablets or capsules, for the treatment of patients whose fibrinolytic 2

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Increase capacity and prevent cardiovascular diseases and to prevent endotoxin effects such as in septic shock to prevent.

The subject matter of the invention is further characterized in claims 2 to 5.

Examples

The following materials and methods were used in all examples.

Chemically pure Notoginsenoside R1 (NR1) was purchased from the National Institute for the Control of Pharmaceuti-cal and Biological Products (Beijing, China). NR1 is a substance with the following formula:

NR1 was dissolved in incubation medium and diluted to reach a final concentration of 0.01 to 100 µg / ml. Lipopolysaccharide (Escherechia coli lipopolysaccharide, serotype 026: B6 prepared by phenol extraction was obtained from Sigma (St. Louis, MO, USA). A solution with the concentration of 1 mg / ml in distilled water was stored at -70 ° C. Morpholinopropanesulfonic acid ( Serva, Germany), guanidine thiocyanate (Fluka, Switzerland), piperazine-N, N'-bis [2-ethanesulfonic acid] (PIPES; Sigma), Seakem LE Agarose (FMC Bioproducts, ME, USA), dCTP [Aloha-32P ] (ICN Radiochemicals, CA, USA) were obtained from the companies mentioned, and the other materials described in the methods are detailed in the relevant citations.

Cell culture

Endothelial cells were isolated from fresh human umbilical cord veins with collagenase (Sigma), similar to a protocol developed by Jaffe et al. Collaborator, J Clin Invest 1973; 52: 2745-56. Cells from 4-6 umbilical cords were pooled and seeded in 75 cm2 cell culture bottles (Costar, MA, USA), which were coated with 1% gelatin from calf skin (Sigma). The cells were mixed to confluence at 37 ° C. in a water vapor-saturated atmosphere of 95% air and 5% CO 2 in medium 199 (Sigma) with 20% heat-inactivated supplemented calf serum (SCS; Hyclone, UT, USA), 100 μg / ml streptomycin , 100 lU / ml penicillin, 250 ng / ml fungizone, 1 mM glutamine (JHR Biosciences, KS, 3rd

AT 402 890 B USA) 2 lU / ml heparin (Liquemin Roche; Hoffmann La Roche, Switzerland), 50 µg / ml ECGS (Technoclone, Austria). The endothelial character of the cells was characterized by their typical cobblestone morphology, by positive immunofluorescence with anti-Von Willebrandt factor VIII antibodies and by the intake of acetylated low density lipoprotein (LDL). Primary cultures were harvested at the time of confluence with 0.05% trypsin / 0.02% EDTA (JRH Biosciences) and sown in a split ratio of 1: 3 in 75 cm2 cell culture bottles. Subconfluent cells were cultured under the same conditions until confluence was reached and were harvested with trypsin / EDTA during exponential cell growth and frozen in 1 ml portions in medium 199 with 10% dimethyl sulfoxide (DMSO) in liquid nitrogen. For the experiments, the cells were thawed at 37 ° C. and cultivated in 6-well plates (diameter 3.5 cm; Costar) in medium 199 mixed with SCS, ECGS and heparin in the concentrations mentioned above until confluence was reached. The average cell densities at confluence were 6x10 * cells / cm2. Cells between the 2nd and 3rd passages were used in all experiments. The cells were always fed with fresh medium the day before the experiments. All materials used in the cell culture were free of endotoxin as determined by the Coatest Endotoxin Kit (Kabi Vitrum, Sweden) (detection limit of the test 5 pg / ml).

Preparation of the conditioned medium (CM) and the extracellular matrix (ECM)

Confluent cultures were rinsed twice with Hank's Balanced Salt Solution (HBSS; Sigma) and mixed at 37 ° C with 1 ml / well of medium 199 with 1.25% SCS and 50 µg / ml ECGS and with NR1 in the listed Concentrations incubated. After the incubation, the cell culture supernatant was collected and after centrifugation - to remove cell debris - stored at -70 ° C. until use. The total number of cells in the corresponding cultures was determined using the counting chamber after trypsinization. ECM from these or similarly treated cultures was determined by the method of Mimuro et al. Co-worker, Blood 1987; 70: 721-28, prepared. The monolayers were washed 3 times with cold phosphate-buffered saline (PBS: 0.01 M sodium phosphate, 0.14 M NaCl, pH 7.4) and the cellular components were mixed with PBS by incubation at 37 ° C. for 10 minutes, 5% Triton X100 extracted. The plates were washed once more with distilled water to remove remaining cellular components and then examined for the presence of cell debris by microscopic examination. This extraction method completely removed visible cell components from the plates and the ECM was extracted by scraping into 1 ml of PBS with 0.1% SDS after 30 minutes of incubation at 37 ° C. The extracts were dialyzed overnight at 4 "C against PBS.

Tests for tPA antigen, uPA antigen, PAI-1 antigen, PAI-1 activity and tPA PAI-1 complexes in CM, in the ECM and in plasma tPA antigen, uPA antigen, PAI-1 antigen and the concentration of tPA PAI-1 Complexes were determined using specific commercially available Enzyme Linked Immunosorbent Assays (ELISAs) (Technoclone) according to the instructions provided by the manufacturer. The test ranges for these tests are between 0.3 and 2.5 ng / ml for tPA, between 0.6 and 10 ng / ml for uPA, between 1.0 and 30 ng / ml for PAI and between 0 for tPA and PAI-1 complexes. 2 and 20 ng / ml. The tPA ELISA determines free tPA and tPA in complex with PAI-1. The uPA ELISA determines free uPA and uPA in complex with PAI-1. The PAI-1 ELISA measures free, complexed and latent PAI-1. The tPA PAI-1 complex ELISA measures only tPA PAI-1 complexes. PAI-1 activity was determined in the plasma samples and in the CM according to the manufacturer's instructions using a titration assay (Technoclone).

Determination of the functional activity of tPA and PAI-1

The activity of tPA and PAI-1 was analyzed after sodium dodecyl sulfate polyacrylamide gel ectrophoresis (SDS-PAGE) using fibrinautography (FA) and reverse fibrinautography (RFA). SDS polyacrylamide gels and buffers were made according to the protocol of Laemmli, Nature 1970; 227: 680-85. FA was developed by Granelli-Piperno u. Collaborator, J Exp Med 1978; 148: 223-34. 100 μl of the corresponding samples were applied to gels consisting of a 10 cm long separating gel with 10% acrylic amide and from a 2 cm long upper gel with 4% acrylic amide and run at room temperature for 16 hours or until the color front reached the lower edge of the gel. After electrophoresis, the gels were first placed in 250 ml of water mixed with 2.5% Triton XI00 (Serva) for 90 minutes (the Triton solution was changed after 45 minutes) to neutralize the SDS, then the gels were placed on a fibrin agar -Indicator film mixed with 1.5% agarose type L (Behring, Germany), 2 mg / ml plasmino- 4

AT 402 890 B gene rich fibrinogen (Organon Teknika, Holland) and 0.2 lU / ml bovine thrombin (Sigma). The gels were incubated at 37 ° C. in a humid chamber and photographed at different times. XRF was carried out by placing the gels on a fibrin film which was basically produced as described above but additionally contained 0.4 lU / ml urokinase (Technoclone). The quantification of tPA and PAI-1 activity in a particular sample was achieved by photographing both lysis zones and lysis-resistant zones on the indicator film. These zones were recorded on tracing paper and the drawn areas were cut out and weighed on an analytical balance.

In order to immunologically identify the plasminogen activator contained in the CM of HUVECs, samples of the CM were incubated at 4 ° C for 24 hours with monoclonal anti-tPA antibodies (MPW 3 VPA; Technoclone) bound to either CNBr-activated Sepharose or monoclonal anti-uPA antibodies (MPW 5 UK; Technoclone) or as control with Sepharose 4B (Pharmacia, Sweden). The Sepharose was then removed by centrifugation and 100 μl of the corresponding sample was analyzed using the SDS-PAGE described above and subsequent FA.

Preparation of the cell lysates and measurement of the TF activity HUVECs were incubated for 6 hours at 37 * C in medium 199 with LPS and / or NR1. The cells were washed 3 times with clotting buffer (130 mM NaCl, 8 mM Na barbital and 12 mM Na acetate, pH = 7.4) and taken up in 300 μl clotting buffer by scraping. The scraped cells were frozen and thawed 3 times. The cell lysates were analyzed for TF activity in a 1-step clotting assay. 100 μl of the cell lysates were incubated with 100 μl of 20 mM CaCl 2 at 37 ° C. for 5 minutes in preheated plastic tubes in a coagulometer (H. Amelung GmbH., Germany). The coagulation was triggered by adding 100 μl of preheated normal human citrate plasma or by adding factor X deficient plasma (Sigma). TF activity was quantified using a standard log-log plot constructed with rabbit brain thromboplastin (Sigma). 100 mU activity was defined as a clotting time of 20 seconds in a standard test with normal human plasma. The coagulatory activity observed corresponds to the TF activity, since no procoagulatory activity of the endothelial cells was found when factor X deficient plasma was used instead of normal plasma.

Quantification of tPA, PAI-1 and TF mRNA levels by Northern blot analysis

The total cellular RNA from endothelial cells was determined using the method of Chomzynski and Sacchi, Anal Biochem 1987; 162: 156-9, described acidic guanidine thiocyanate-phenol-chloroform extraction. The RNA precipitate was resuspended in 50 μl of 0.5% SDS and the concentration was determined at 260 nM. For the Northern blot analysis, the RNA samples were electrophoresed in a 1.2% agarose gel and then the fractionated RNA was transferred to a Duralon-UV ™ membrane (Stratagene, CA, USA) by capillary action. The RNA blots were sealed in seal-a-meal bags and prehybridized in 50 mM PIPES, 100 mM NaCl 50 mM NaPhosphate 1 mM EDTA, mixed with 5% SDS for at least 3 hours at 57 ° C. The prehybridization buffer was discarded and mixed with fresh prehybridization buffer with 106 CPM / ml of the 32 P labeled cDNA probes for either human tPA, human PAI-1, human TF or rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Hybridization was carried out in a water bath at 57C overnight. After hybridization, blots were removed from the sachet and washed for 10 minutes in 100 ml of 5% SDS, 0.2xSSC at room temperature. The blots were then washed for 20 minutes in 400 ml of 5% SSC, 1x SSC at the hybridization temperature. After hybridization, RNA blots were air dried and XAR-5 X-ray films were exposed at -70 ° C (Eastman Kodak, NY, USA). To quantify the differences in the specific mRNA expression, the developed films were scanned with a densitometer (Hirschmann Eiscript 400; Hirschmann, Germany). The scanned data for each specific mRNA was compared to the intensity of the GAPDH mRNA. cDNA samples

The following cDNA fragments were used as samples in the hybridization experiments: a 1.5 kb Sma 1 Hind III fragment of human t-PA cDNA, a 1.4 kb EcoRI / Bg1 II fragment of human PAI-1 cDNA of the 3.2 kb transcript, 0, 64 kb EcoRI / 'Ecol fragment of human TF cDNA and a 1.2 kb PstI fragment of a rat GAPDH cDNA, which was used as an internal standard probe. The cDNA fragments were radioactive with a Random Prime DNA Labeling Kit (Boehringer Mannheim, Germany) marked. 5

AT 402 890 B

Animal experiments

Male Balb C mice (18-30 g body weight) were used in this study. All experiments were carried out under etheric anesthesia. The mice were injected intranvenously via the tail vein with LPS (10 ng / g) and / or NR 1 (1 µg / g) in a volume of 5 µg / l. Blood samples anticoagulated with sodium citrate (0.13 M final concentration) were obtained at the indicated times. Platelet-free plasma was obtained by centrifugation at 2500 g for 30 minutes at 4'C and stored at -70 ° C until testing.

Statistical analysis

The results are given as means £ standard deviation. An unpaired Student's t-test was used to determine the significance.

Example 1: Effect of NR 1 on the production of tPA, uPA and PAI-1 in cultured human umbilical vein endothelial cells

Effect of Notoginsenoside R 1 (NR 1) on tPA antigen (panel A), t-PA PAI-1 complexes (panel B) and PAI-1 antigen (panel C) production in cultured HUVECs (Fig. 1). HUVECs were used for 24 Incubated for hours with different concentrations of NR 1 (0.01-100 µg / ml) and the CM was examined for tPA antigen, PAI-1 antigen and tPA PAI-1 complexes as described under Materials and Methods. The results are the mean values of 3 experiments, each carried out in triplicate. The values are as averages £ S.D. shown in Fig. 1. Significances are given compared to the control (* p <0.05; ”p <0.01; *” p <0.001). As shown in FIG. 1A, the treatment of HUVECs with increasing concentration of NR1 for 24 hours led to a dose-dependent increase in the tPA antigen in the CM of cells treated in this way: maximum effects were achieved with 100 μg / ml NR 1 (100 mg / ml NR 1: 9.6 pounds 0.7 ng / 105 cells / 24h; control: 5.8t 0.4 ng / 105 / 24h; n = 9, p <0.05) reached. As shown in Fig. 1B, the tPA PAI-1 complexes in the CM also increased similarly in the presence of increasing concentrations of NR 1 (100 µg / ml NR 1: 63.5 ± 2.6 ng / 105 cells / 24h; Control: 40.2 pounds 7ng / 105 cells / 24h; n = 9, p <0.01.

Effect of Notoginsenoside (NR 1) on PAI-1 antigen production in the ECM of cultured HUVECs: (Fig. 2) HUVECs were incubated for 24 hours with different concentrations of NR 1 (0.01-100ug / ml). The ECM was obtained as described under Material and Methods and examined for PAI-1 antigen. The values represent averages £ S.D. from 6 independent wells. PAI-1 antigen in the CM and in the ECM of HUVECs treated with NR 1 was not significantly changed compared to the controls (CM: 100 μg NR 1 / ml: 2.92 pounds 0.32 μg / 105 Cells / 24h; control: 2.78 lb. 0.45 µg / 105 cells / 24h; n = 9 .ECM: lOOug / mlNR 1: 42.55 lb. 3.15 ng / ml / 24h; control: 42.27 lb. 1.66 ng / ml / 24 h; n = 6) (Fig. 1C, Fig. 2).

Time course of the tPA antigen (field A) and PAI-1 antigen (field B) Production of cultured HUVECs after treatment with Notoginsenoside R1 (NR 1): (Fig. 3) HUVECs were absent (open circles) or present for the indicated periods of 100ug / ml NR 1 (full circles). At the specified times, the corresponding CM was harvested and tested for tPA antigen and PAI-1 antigen as described under Materials and Methods. The results represent the mean values of 3 experiments, each carried out in a triple arrangement. The values are as mean values £ S.D., ’p <0.05; “P <0.01 compared to control. As shown in Fig. 3, the tPA antigen in the CM of HUVECs treated with 100 µg / ml NR 1 for 6, 12 or 24 hours increased time-dependent compared to the control, whereas the PAI-1 antigen in the CM of this type increased treated cells did not change significantly. 6

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Notoginsenoside R 1 influences the uPA antigen secretion of cultivated HUVECs

When the CM of HUVECs incubated in the presence of 100 µg / ml NR 1 was examined for uPA antigen, there was a significant change in the amount of uPA antigen produced by these cells compared to the CM of HUVECs under control conditions had been cultured (100 µg / ml NR 1: 2.9 pounds 06 ng / 106 cells / 24h; control: 2.5 ± 0.8 ng / 106 cells / 24h; n = 9).

Example 2: Effect of NR 1 on tPA and PAI-1 activity in cultured human umbilical vein endothelial cells

Notoginsenoside R1 increases tPA activity and reduces PAI-1 activity in cultured HUVECs. Fibrin Autography (FA) of HUVECs CM samples: (Fig. 4) CM of HUVECs was collected after 24 hours and examined with SDS PAGE and subsequent FA as described under Material and Methods. Lane 1: untreated CM from HUVECs: Lane 2: CM from HUVECs preincubated with anti-tPA monoclonal antibodies coupled to Sepharose; Lane 3: CM from HUVECs preincubated with anti-uPA monoclonal antibodies coupled to Sepharose: Lane 4: CM from HUVECs preincubated with Sepharose 4B; Lane 5: purified human tPA; Lane 6: purified human uPA. When CM obtained from HUVECs incubated for 24 hours under control conditions was analyzed with SDS PAGE and subsequent FA, 2 dominant lysis zones with an apparent molecular weight of 70,000 and 120,000 were found. These lysis zones could be removed by preincubation with monoclonal anti-tPA antibodies but not by preincubation with monoclonal anti-uPA antibodies (FIG. 4). It can therefore be concluded that the lysis zone at 70 kDa was caused by free tPA and that the high molecular lysis zone was derived from tPA in complex with PAI-1.

Effect of Notoginsenoside R1 (NR1) on tPA activity and on activity associated with the tPA PAI-1 complex (field A) and on PAI-1 activity (field B) in cultured HUVECs after analysis with fibrin autography (FA) and reversed Fibrin Autography (RFA): (Fig. 5)

After the incubation of confluent HUVECs for 24 hours with different concentrations of NR 1 (0.001-100ug / ml), the CM was processed with SDS PAGE and subsequent FA or RFA as described under Materials and Methods. The lysis zones and the lysis-resistant zones in these figures were transferred to tracing paper, cut out and weighed on an analytical balance. The weights of these tracing papers were plotted against the concentrations of NR 1 (lower panel). The data represent results from one of 3 independent experiments that showed similar results. Lane 1: Control; Lane 2: 0.01 µg / ml NR 1; Lane 3: 0.1ug / ml NR 1; Lane 4: 1.0ug / ml NR 1; Lane 5: 10 µg / ml NR 1; Lane 6: 100 / ml NR 1. If the CM of HUVECs, which were cultured without or with increasing concentrations of NR 1 for 24 hours, was analyzed with FA or RFA, a dose-dependent increase in the size of the lysis zones could be determined, where, on the other hand, the size of the lysis-resistant zones decreased with increasing amounts of NR 1 (FIGS. 5A and B). If the size of the lysis zones or the lysis-resistant zones was quantified as described in the materials and methods, an up to 3-fold increase in tPA-dependent lysis could be determined, whereas the PAI-1-dependent lysis resistance to 20% in Presence of 100ug / ml NR 1 decreased compared to the control (Fig. 5A and B).

Example 3: Effect of NR 1 on the tPA and PAI-1 mRNA in cultured human umbilical vein endothelial cells

Effect of Notoginsenoside R 1 (NR 1) on tPA and PAI-1 mRNA Expression in HUVECs: (Fig. 6)

Confluent HUVECs were incubated for 12 hours in the absence (C) or in the presence (T) of NR 1- (100ug / ml). Northern blot analysis of the RNA extracts from untreated and NR 1-treated HUVECs was carried out using 32P-labeled cDNA probes for tPA, PAI-1 and GAPDH mRNA. The intensity of the bands present on the autoradiogram was evaluated with the aid of densitometry and the specific mRNA for tPA or PAI-1 was normalized against GAPDH mRNA in order to take differences in the loading into account. The signal intensity was compared as the ratio of the signal from HUVECs treated with NR 1 to the signal from untreated 7

Claims (5)

AT 402 890 B control cells. These data represent results from 2 independent experiments that showed similar results. As shown in FIG. 6, the stimulating effect of NR 1 on the secretion of tPA in HUVECs was also reflected at the level of the specific mRNA expression. The tPA-specific mRNA increased up to a 2-fold value in HUVECs treated with 100ug / ml NR1 for 12 hours, whereas the PAI-1-specific mRNA expression was not regulated by NR 1 (3.2 kb: 82% of the control , 2.2kb: 86% of the control). When Northern blot experiments were performed in the presence of 10ug / ml cycloheximide, the stimulating effect of NR 1 on the tPA specific mRNA was prevented (data not shown). Example 4: Effects of NR 1 on endotoxin-induced upregulation of PAI-1 antigen, activity and PAI-1 mRNA in vitro. As shown in Fig. 7, the upregulation of the PAI-1 antigen after treatment of the cells with LPS (1 µg / ml) for 12 hours is antagonized by simultaneous treatment with different concentrations of NR 1. The extent of the antagonism was dose-dependent in relation to the NR 1 concentration (0.100 μg / ml) and the increase in the PAI-1 antigen induced by LPS was significantly reduced by the coincubation of the cells with 100 μg / ml NR 1 (control cells: 347 ± 34ng / l05 cells / I2h, LPS treated cells: 946t42ng / 105 cells / 12h, LPS and NR 1 treated cells: 469 ± 29ng / 105 cells / 12h). The change in the PAI-1 activity of the cells was parallel to the change in the PAI-1 antigen (control cells: 5.48 ± 0.78U / 105 cells / 12h, LPS treated cells: 8.22 ± 0.18U / 105 / cells / 12h , LPS and NR 1 treated cells: 4.77 ± 0.26U / 105 cells / 12h, n = 6). The mRNA for PAI-1 was measured in cells treated with a u.g / ml LPS and / or 100ug / ml NR 1. The increase induced by LPS to twice the amount of PAI-1-specific mRNA (3.2 kb) was reduced to a 1.37-fold increase in the presence of both LPS and NR 1 (FIG. 8). Example 5: Effects of NR 1 on LPS-induced upregulation of PAI-1 activity in vivo. In vivo studies showed that injection of LPS in mice resulted in a rapid increase in plasma PAI-1 activity. With an LPS dose of 10 ng / g (body mass), a significant up to 7-fold increase over the control value could be determined 2 hours after the injection, while maximum values were reached 4 hours after the injection. At later times, the PAI-1 activity gradually returned to normal. In contrast, PAI-1 activity in animals treated simultaneously with LPS and NR 1 (1ug / g) returned to the control values 4 hours after the injection (LPS-treated group: 11.3i3.1U / ml, LPS and NR 1 treated Group: 4.3 ± 1.0U / ml, control group 4.9iO, 3U / ml, n = 5-8) (Fig. 9). Example 6: Effects of NR 1 on the induction of TF activity and mRNA caused by endotoxin (LPS) in cultivated HUVECs: Only a very small amount of TF activity was found in untreated HUVECs (0.78 * 0.15 mU / 106 cells, n = 9). The TF activity increased in HUVECs after treatment with LPS (lug / ml for 6h) to a value of 88.6i6.5mU / 106 cells (n = 6). The TF activity in HUVECs measured after 6 hours of treatment with mg / rnl LPS was also significantly antagonized by coincubation with NR 1 (LPS and NR 1 treated cells: 56.0 * 1.9 mU / 106 cells). The extent of the antagonism was also dose-dependent in relation to the NR 1 concentration, with about 36.8% inhibition being achieved with 100 μg / ml MR 1 (FIG. 10). A significant increase in TF mRNA was observed after treatment of HUVECs with LPS, which reached a 9-fold (2.4 * 3.1 ± 3.5 kb) increase over control values after 2 hours. The TF mRNA values increased by LPS were antagonized in a similar manner by coincubation with MR 1; the TF mRNA was reduced to a value approximately 4 times higher than the control. Treatment of the cells with 100 μg / ml NR 1 alone reduced the TF mRNA values to 40% of the control values (FIG. 11). Claims 1. Use of Notoginsenoside R1 (NR1) for the manufacture of medicaments for the treatment of conditions with reduced fibrinolytic activity or for the inhibition of endotoxin effects. 8 AT 402 890 B 2. Use of Notoginsenoside R1 (NR1) for the manufacture of medicaments for the treatment of patients and animals under endotoxin shock or for the prevention of endotoxin shock according to claim 1. 3. Use of Notoginsenoside R1 (NR1) for the production of medicaments for the prophylactic or therapeutic treatment of patients with coronary heart disease, peripheral artery disease and of patients who suffer from myocardial infarction or angina pectoris in order to increase their fibrinolytic potential, according to claim 1. 4. Use of Notoginsenoside R1 (NR1) for the production of medicaments for the prevention of conditions with reduced fibrinolytic activity or of endotoxin effects in healthy persons according to claim 1, 5. Use of Notoginsenoside R1 (NR1) for the production of medicaments in the form of solutions, 75 tablets or capsules in the presence of additives, stabilizers or substances which increase the bioavailability, according to any one of claims 1 to 4. Including 10 sheets of drawings 20 25 30 35 40 45 50 9 55